Regulation of Coronaviral Poly(A) Tail Length during Infection

Abstract

The positive-strand coronavirus genome of ~30 kilobase in length and subgenomic (sg) mRNAs of shorter lengths, are 5’ and 3’-co-terminal by virtue of a common 5’-capped leader and a common 3’-polyadenylated untranslated region. Here, by ligating head-to-tail viral RNAs from bovine coronavirus-infected cells and sequencing across the ligated junctions, it was learned that at the time of peak viral RNA synthesis [6 hours postinfection (hpi)] the 3’ poly(A) tail on genomic and sgmRNAs is ~65 nucleotides (nt) in length. Surprisingly, this length was found to vary throughout infection from ~45 nt immediately after virus entry (at 0 to 4 hpi) to ~65 nt later on (at 6 h to 9 hpi) and from ~65 nt (at 6 h to 9 hpi) to ~30 nt (at 120-144 hpi). With the same method, poly(U) sequences of the same lengths were simultaneously found on the ligated viral negative-strand RNAs. Functional analyses of poly(A) tail length on specific viral RNA species, furthermore, revealed that translation, in vivo, of RNAs with the longer poly(A) tail was enhanced over those with the shorter poly(A). Although the mechanisms by which the tail lengths vary is unknown, experimental results together suggest that the length of the poly(A) and poly(U) tails is regulated. One potential function of regulated poly(A) tail length might be that for the coronavirus genome a longer poly(A) favors translation. The regulation of coronavirus translation by poly(A) tail length resembles that during embryonal development suggesting there may be mechanistic parallels.

Interplay between the Poly(A) Tail, Poly(A)-Binding Protein, and Coronavirus Nucleocapsid Protein Regulates Gene Expression of Coronavirus and the Host Cell

“…In coronaviruses, however, the mechanisms by which the two processes are regulated remain unclear. Based on the results from the current study and others, (i) the poly(A) tail can be bound by PABP and function in translation (46), (ii) the poly(A) tail is a start site for negative-strand RNA synthesis (47), (iii) the poly(A) tail can also be bound by the N protein with high affinity (Fig. 1), (iv) the N protein can interact with viral replicase proteins (8)(9)(10)(11)(12)(13)(14) and nsp9 ( Fig.…”

Coronavirus cis-Acting RNA Elements

“…This requirement corresponds well to the minimal binding site of the poly(A)-binding protein (PABP) on DI RNAs poly(A) sequences (Spagnolo and Hogue, 2000). Recent studies further suggest that 3 0 poly(A) tail lengths may vary between 30 and 65 nt in the course of viral replication in vitro (Wu et al, 2013) as was shown for both beta-and gammacoronavirus infections and in a range of cell types, both in vitro and in vivo (Shien et al, 2014). The biological significance of these observations is currently unclear.…”

“…The resulting set of 3 0 antileadercontaining sg minus-strand RNAs is subsequently used as templates for the production of the characteristic nested set of 5 0 leader-containing mRNAs in coronavirus-infected cells (Lai et al, 1983;Sawicki and Sawicki, 1995;Sawicki et al, 2001;Sethna et al, 1989;Spaan et al, 1983). Sg minus-strand RNAs contain a U-stretch at their 5 0 end, providing a possible template for 3 0 polyadenylation of sg mRNAs Wu et al, 2013).…”

RNA structure analysis of alphacoronavirus terminal genome regions

“…This requirement corresponds well to the minimal binding site of the poly(A)-binding protein (PABP) on DI RNAs poly(A) sequences (Spagnolo and Hogue, 2000). Recent studies further suggest that, in the course of BCoV infection, 3 poly(A) tail lengths vary between 30 and 65 nts (Wu et al, 2013). This poly(A) tail length variation was confirmed to occur in beta-and gammacoronavirus infections and in a range of cell types, both in vitro and in vivo (Shien et al, 2014).…”

“…The set of 3 antileader-containing sg minus-strand RNAs is subsequently used as templates for the production of the characteristic nested set of 5 capped, 5 leader-containing and 3 -polyadenylated sg mRNAs in coronavirus-infected cells (Lai et al, 1983;Sawicki et al, 2001;Sawicki and Sawicki, 1995;Sethna et al, 1989;Spaan et al, 1983). Sg minus-strand RNAs contain a U-stretch at their 5 end, thus providing a template for 3 polyadenylation of sg mRNAs Wu et al, 2013).…”